This invention is in the field of detection of fluorescence polarization, e.g., in microfluidic devices.
Detection of single nucleotide polymorphisms (SNPs) and other genetic phenomena is an increasingly important technique in molecular biology and medicine. For example, in medical contexts, polymorphism detection is useful for diagnosing inherited diseases and susceptibility to diseases. The detection of SNPs and other polymorphisms can also serve as a basis for tailoring or targeting treatment, i.e., where certain allelic forms of a polymorphism are associated with a response to a particular treatment. In molecular biology, polymorphism detection is fundamental in a variety of contexts, including molecular marker assisted breeding (e.g., of important crop varieties such as Zea and other Graminea, soybeans, etc.), the study of gene diversity, gene regulation and other genetic, epigenetic or para-genetic phenomena.
Many techniques exist for measuring nucleic acid hybridization for polymorphism detection, as well as for other purposes. In addition to standard Southern and northern blotting, complex arrays of nucleic acid probes are available from a variety of commercial sources, as are solution based detection methods such as those utilizing fluorescence resonance energy transfer (FRET), molecular beacons, or other real-time solution-based hybridization detection methods. These hybridization methodologies typically involve the use of one or more probe, e.g., which includes a fluorophore or other label. Specific hybridization is detected by localization of probe label signals in solid phase hybridization methods such as Southern blotting, or array-based versions thereof, or by real time optical and/or spectroscopic methods which monitor changes in fluorescence in solution, e.g., as detected by FRET.
One additional technique has recently been used for detecting hybridization formation between nucleic acids, e.g., in the presence of polylysine. As described by the inventors in Nikiforov and Jeong xe2x80x9cDetection of Hybrid Formation between Peptide Nucleic Acids and DNA by Fluorescence Polarization in the Presence of Polylysinexe2x80x9d (1999) Analytical Biochemistry 275:248-253, Fluorescence Polarization (FP) provides a useful method to detect hybridization formation between nucleic acids. This method is applicable to hybridization detection, e.g., to monitor SNPs.
Generally, FP operates by monitoring the speed of rotation of fluorescent labels, such as fluorescent dyes, e.g., before, during and/or after binding events between probes which comprise the labels and target molecules. In short, binding of the probe to a target molecule ordinarily results in a decrease in the speed of rotation of the bound probe, resulting in a change in FP.
For example, when a fluorescent molecule is excited by a polarized light source, the molecule will emit fluorescent light in a fixed plane; that is, the emitted light is also polarized, provided that the molecule is fixed in space. However, because the molecule is typically rotating and tumbling in space, the plane in which the fluoresced light is emitted varies with the rotation of the molecule (also termed the rotational diffusion of the molecule). Restated, the emitted fluorescence is generally depolarized. The faster the molecule rotates in solution, the more depolarized it is. Conversely, the slower the molecule rotates in solution, the less depolarized, or the more polarized it is. The polarization value (P) for a given molecule is proportional to the molecule""s xe2x80x9crotational correlation time,xe2x80x9d or the amount of time it takes the molecule to rotate through an angle of 57.3xc2x0 (1 radian). The smaller the rotational correlation time, the faster the molecule rotates, and the less polarization will be observed. The larger the rotational correlation time, the slower the molecule rotates, and the more polarization will be observed. Rotational relaxation time is related to viscosity (xcex7), absolute temperature (T), molar volume (V), and the gas constant (R). The rotational correlation time is generally calculated according to the following formula:
Rotational Correlation Time=3xcex7V/RTxe2x80x83xe2x80x83(1) 
As can be seen from the above equation, if temperature and viscosity are maintained constant, then the rotational relaxation time, and, therefore, the polarization value, is directly related to the molecular volume. Accordingly, the larger the molecule, the higher its fluorescent polarization value, and conversely, the smaller the molecule, the smaller its fluorescent polarization value.
In the performance of fluorescent binding assays, a typically small, fluorescently labeled molecule, e.g., a ligand, antigen, etc., having a relatively fast rotational correlation time, is used to bind to a much larger molecule, e.g., a receptor protein, antibody etc., which has a much slower rotational correlation time. The binding of the small labeled molecule to the larger molecule significantly increases the rotational correlation time (decreases the amount of rotation) of the labeled species, namely the labeled complex over that of the free unbound labeled molecule. This has a corresponding effect on the level of polarization that is detectable. Specifically, the labeled complex presents much higher fluorescence polarization than the unbound, labeled molecule.
Generally, the fluorescence polarization level is calculated using the following formula:
P=[I(∥)xe2x88x92I(xe2x8axa5)]/[I(∥)+I(xe2x8axa5)]xe2x80x83xe2x80x83(2) 
Where I(∥) is the fluorescence detected in the plane parallel to the excitation light, and I(xe2x8axa5) is the fluorescence detected in the plane perpendicular to the excitation light.
In addition to Nikiforov and Jeong (1999), above, other references which discuss fluorescence polarization and/or its use in molecular biology include Perrin (1926). xe2x80x9cPolarization de la lumiere de fluorescence. Vie moyenne de molecules dans l""etat excite.xe2x80x9d J Phys Radium 7, 390; Weber (1953) xe2x80x9cRotational Brownian motion and polarization of the fluorescence of solutionsxe2x80x9d Adv Protein Chem 8, 415; Weber (1956). J Opt Soc Am 46, 962; Dandliker and Feigen (1961), xe2x80x9cQuantification of the antigen-antibody reaction by the polarization of fluorescencexe2x80x9d Biochem Biophys Res Commun 5, 299; Dandliker and de Saussure (1970) (Review Article) xe2x80x9cFluorescence polarization in immunochemistryxe2x80x9d Immunochemistry 7, 799; Dandliker W B, et al. (1973). xe2x80x9cFluorescence polarization immunoassay. Theory and experimental method.xe2x80x9d Immunochemistry 10, 219; Levison S A, et al. (1976), xe2x80x9cFluorescence polarization measurement of the hormone-binding site interactionxe2x80x9d Endocrinology 99, 1129; Jiskoot et al. (1991), xe2x80x9cPreparation and application of a fluorescein-labeled peptide for determining the affinity constant of a monoclonal antibody-hapten complex by fluorescence polarizationxe2x80x9d Anal Biochem 196, 421; Wei and Herron (1993), xe2x80x9cUse of synthetic peptides as tracer antigens in fluorescence polarization immunoassays of high molecular weight analytesxe2x80x9d Anal Chem 65, 3372; Devlin et al. (1993), xe2x80x9cHomogeneous detection of nucleic acids by transient-state polarized fluorescencexe2x80x9d Clin Chem 39, 1939; Murakami et al. (1991), Fluorescent-labeled oligonucleotide probes detection of hybrid formation in solution by fluorescence polarization spectroscopy.xe2x80x9d Nuc. Acids Res 19, 4097. Checovich et al. (1995), xe2x80x9cFluorescence polarization-a new tool for cell and molecular biologyxe2x80x9d (product review), Nature 375, 354-256; Kumke et al. (1995), xe2x80x9cHybridization of fluorescein-labeled DNA oligomers detected by fluorescence anisotropy with protein binding enhancementxe2x80x9d Anal Chem 67:21, 3945-3951; and Walker et al. (1996), xe2x80x9cStrand displacement amplification (SDA) and transient-state fluorescence polarization detection of mycobacterium tuberculosis DNAxe2x80x9d Clinical Chemistry 42:1, 9-13.
One difficulty in the use of FP to monitor hybridization of nucleic acids is that the change in FP which occurs simply upon binding of a labeled probe to a complementary nucleic acid has previously been observed to be small. Thus, helper molecules such as DNA binding proteins or polycations are used to increase the change in FP (and, therefore, the dynamic range of the assay) which is observed upon hybridization of nucleic acids (e.g., by binding to the hybridized nucleic acid, thereby increasing the size of the complex). While increasing the dynamic range of the assay, this approach also increases the complexity of the assay and secondary effects caused by helper molecules can bias the assay.
Quite surprisingly, the present invention overcomes these previous difficulties, providing a robust assay for direct detection of nucleic acid hybridization by monitoring changes in FP.
It has, quite surprisingly, been discovered that the use of neutral or positively charged fluorescent labels on nucleic acid probes results in a relatively large change in observed FP of the probe label during nucleic acid hybridization. Thus, probes (e.g., PNAs, DNAs, LNAs, RNAs or other nucleic acids, or even other nucleic acid binding moieties) can be labeled with neutral or positively charged fluorescent dyes such as rhodamine or BODIPY and FP can effectively be used to monitor hybridization of such labeled probes to target nucleic acids. This surprising discovery provides the basis for simplified and less biased FP assays than those used in the past.
Accordingly, the present invention provides methods of performing nucleic acid hybridization analysis (i.e., using probes comprising neutral or positively charged fluorescent dyes). This analysis is useful, e.g., for polymorphism detection, as well as for many other applications.
In addition to providing new methods, the present invention provides assay systems, kits, computer implemented processes and microfluidic systems for practicing the methods of the invention. For example, assay systems with containers comprising probes comprising neutral or positively charged fluorescent dyes are a feature of the present invention, e.g., in combination with apparatus for performing FP measurements.